Losa et al: Somatoliberin-like immunoreactivity in human seminal plasma: RIA interference 367 J. Clin. Chem. Clin. Biochem.
Vol. 26, 1988, pp. 367-371
© 1988 Walter de Gruyter & Co.
Berlin · New York
Evidence for a Nonspecific Factor Interfering in the Radioimmunoassay of Somatoliberin-Like Immunoreactivity in Human Seminal Plasma
By M. Losa, G. Wolfram, /. Schopohl, S. Sobieszczyk1) and K. von Werder
Medizinische Klinik Innenstadt, University of Munich, Federal Republic of Germany
(Received July 27, 1987/February 15, 1988)
Summary: In the present study we investigated the nature of somatoliberin-like immunoreactivity in human seminal plasma. In unextracted seminal plasma, somatoliberin-like immuno reactivity represented 3.8 §/1 somatoliberiii, with a dilution curve that ran almost completely parallel to that of synthetic somatoliberin standard. After extraction by adsorption on hydrophobic CIS Sep-pak cartridges or immunoaffmity chro- matography, no somatoliberin-like immunoreactivity in seminal plasma could be detected. After gel permeation chromatography on a Sephadex G-50 fine column, all somatoliberin-like immunoreactivity of unextracted seminal plasma was recovered at the void volume of the column. When equal volumes of unextracted seminal plasma and radioiodinated somatoliberin were coincubated for two days at 4 °C, gel chromatography revealed the disappearance of the normal [J?5I]somatoliberin peak, replaced by a new peak eluting shortly after the total volume of the column. When unextracted seminal plasma was heated at 90 °C for 10 min or submitted to ultrafiltration, the interfering factor was no longer detectable. Our data show that enzymic degradation of radioiodinated somatoliberin led to misleadingly high concentrations of somatoliberin-like immunoreactivity in seminal plasma. These phenomena should therefore be considered when performing radioimmunoassays of short chain peptides in biological fluids.
Introduction
Somatoliberin was first isolated from two pancreatic islet cell tumours (1,2), and subsequently from normal human hypothalämus (3). Using immunohistochem- ical and radioimmunoassay techniques, somatoliberin has been mainly localized in the infundibular region of the hypothalämus (4). Only picomol quantities of somatoliberiii are present in the hypothalämus (4), but somatoliberin has also been detected in other areas of human brain and gut (5, 6). Occurrence at extrahypothalamic sites has also been reported for other known hypothalamic hypophysiotropic hor- mones (7). Moreover, most hypothalamic hormones have been shown to possess biological activities in- dependent of pituitary regulation (8). Somatoliberin
') Present address: Department of Medicine, Section of En- docrinology, University of Chicago, Illinois, USA.
also has been found to stimulate enzyme secretion of the exocrine pancreas (9).
Peptide hormones have been found in seminal plasma of normal subjects (10), but the physiological signif- icance of this finding is unknown. In the present study we investigated and characterized the presence of im- munoreactive somatoliberin in human seminal plasma.
Materials and Methods Seminal plasma
A pool of seminal plasma was obtained from the department of andrology of the University of Munich. Semen samples were collected into a plastic container by masturbation and were delivered to the laboratory within 2 h. The pool of seminal plasma, obtained by mixing different specimens, was centri- fuged at 1600 g for 10 min and the supernatant was aliquoted into 1 ml plastic vials, which were stored at —20 °C until use.
Semen sample aliquots were thawed at room temperature before recentrifugation at 1600# for 10 min to remove cellular debris.
J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 6
368 Losa et al.: Somatoiiberin-like immunoreactivity in human seminal plasma: RIA interference Somatoliberin radioimmunoassay
Somatoliberin was measured using a specific antiserum raised in rabbits immunized with synthetic Somatoliberin \ —44 cou- pled to bovine thyroglobulin. This antibody is directed against the C-terminal region of Somatoliberin and binds equally, on a equimolar basis, to Somatoliberin 1—44, Somatoliberin 1—40, and Somatoliberin 1—29 (11). Somatoliberin was radioiodi- nated by the chloramine T method and purified by a two step Chromatographie procedure: The tracer was initially applied to a Sephadex G-10 column (1 χ 10 cm; Pharmacia, Uppsala, Sweden) and eluted in acetic acid 0.1 mol/1 + bovine serum albumin 1 g/1. The first peak, containing iodinated proteins, was collected and applied to a Sephadex G-50 fine column (1 χ 56 cm; Pharmacia, Uppsala, Sweden) and eluted with the same buffer as above. The descending part of the second peak, showing the highest binding to Somatoliberin antiserum, was pooled and diluted in radioimmunoassay buffer (Na2HPO4
0.063 mol/1 and Na2EDTA 0.013 mol/1, pH 7.4, containing bovine serum albumin 1 g/1, NaN3 0.2 g/1, Triton X-100 1 ml/1, and aprotinin (Trasylol®, 400 000 Kallikrein inhibitory units/1) at a concentration of about 5000 counts per minute. Somato- liberin tracer was stored frozen at —20 °C until use. Standards, diluted in radioimmunoassay buffer, or unknown samples (0.1 ml) were incubated in polypropylene tubes with 0.1 ml anti- serum, diluted 1: '80 000 in radioimmunoassay buffer, for two days at 4 °C. After addition of Somatoliberin tracer, containing 7 g/1 rabbit γ-globulins, incubation was continued for one day at 4 °C. Antibody-bound and free Somatoliberin were separated by a double antibody procedure, using 0.1 ml goat antirabbit antiserum diluted 1:60 with radioimmunoassay buffer.
Gel chromatography
One-mi samples were separately applied to a Sephadex G-50 fine column (1 χ 56 cm), equilibrated, then eluted with radioim- munoassay buffer. Samples and calibration standards (125I, [usl]somatoliberin, and [12SI]thyroglobulin) were eluted at 4 °C at a flow rate of 15 ml/h. One-mi fractions were collected and kept frozen at —20 °C until assayed or, in case of radioiodinated substances, directly counted in a gamma-counter.
Affinity chromatography of Somatoliberin
The γ-globulin fraction of antiserum K1783 was purified on a Protein-Α Sepharose column (Pharmacia, Uppsala, Sweden).
Absorbed γ-globulins were eluted with acetic acid 0.1 mol/1 and immediately desalted on a Sephadex G-25 column ( 1 x 1 2 cm) equilibrated in Na-borate-HCl 0.01 mol/1 buffer, pH 8.7, γ- globulins were conjugated to 4B-CNBr activated Sepharose (Pharmacia, Uppsala, Sweden) following the-procedure sug- gested by the manufacturer. A 0.5 χ 1 cm column was prepared with the immunoadsorbent and equilibrated with radioimmu- noassay buffer. Samples were diluted 3-fold with radioimmu- noassay buffer and successively applied to the immunoaffinity column. The column was washed with 10 ml radioimmunoassay buffer and the antibody-bound Somatoliberin was eluted with acetic acid 0.5 mol/1 containing bovine serum albumin 1 g/1.
After lyophilization, samples were reconstituted with radioim- munoassay buffer before assay.
Somatoliberin extraction
The extraction method of Frohtnan et al. (12) was used with minor modifications. Briefly, 1 ml of seminal plasma was di- luted with 2 ml trifluoroacetic acid 0.01 mol/1 and applied onto hydrophobic C 18 Sep-pak cartridges (Waters Associates, Mil- lipore Corp., Milford, USA) previously activated by successive applications of trifluoroacetic acid 0.01 mol/1, acetonitrile (vol- ume fraction 0.8) in trifluoroacetic acid 0.01 mol/1, and trifluo- roacetic acid 0.01 mol/1 again. The column was then washed
with 10 ml trifluoroacetic acid 0.01 mol/1 and eluted with acetonitrile (volume fraction 0.8) in trifluoroacetic acid 0.01 mol/1. The eluate was lyophilized and then reconstituted in radioimmunoassay buffer before being assayed.
Results
The detection limit of the assay (defined as the small- est detectable concentration of ligand at 3 standard deviations from 0 hormone concentration) was 50-70 ng/1 and 50% binding occurred at 400 — 500 ng/1. Extraction of radioiodinated Somatoliberin by affinity chromatography or adsorption onto Sep-pak CIS cartridges yielded a mean recovery of 78.3
± 3.5% and 87.1 ± 2.3% (± SE), respectively. Unex- tracted seminal plasma contained 3.8 μ§/1 somatoli- berin like-immunpreactivity at 50% binding of the standard curve. The dilution curve of unextracted seminal plasma ran parallel to the standard curve, though a slight deviation to the left occurred at the highest dilutions (fig. 1). However, no detectable amount of Somatoliberin (< 50 ng/1) was measured after extraction by affinity chromatography or acl·
sorption onto C 18 Sep-pak cartridges. On the other hand, the Somatoliberin immunoadsorbent column and the C18 Sep-pak cartridge did not retain the somatoliberin-like immunoreactivity of seminal plasma, since after adsorption to the column and cartridge elevated levels of somatoliberin-like immu- noreactivity could still be detected (< 2.5 μg/l) in the effluent.
Following gel permeation chromatography, the so- matoliberin-like immunoreactivity in seminal plasma did not coelute with Somatoliberin standard and tracer, but appeared in the void volume of the column (fig. 2). Twenty-five ng Somatoliberin 1 —44 in 0.5 ml
120- 100- 80-
° 60- o
40- 20-
101 102 103 104
α Somatoliberin [ng/l]
L I 1 I .| . . .1 . ..|
1:1281:64 1:16 1:8 K 1:2 1:1 a Seminal plasma dilutions
Fig. 1. Radioimmunoassay of unextracted seminal plasma. Di- lution curves for synthetic Somatoliberin 1 -44 (D-D) and unextracted seminal plasma (β—Β) are shown.
B/Bo, bound to maximum bound ratio.
J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 6
Losa et al.: Somatoliberin-Iike immunoreactivity in human seminal plasma: RIA interference 369
Somatoliberin 1-44
Somatoliberin (ng/l) NJ 4- O OD O N O O O O O Co o o o o o c
l \ A
„__ J \J1JL__
10 20 30 40
Elution volume [ml] 50 60 Fig. 2. Sephadex G-50 fine chroinatography of unextracted
seminal plasma (β—·) and of 25 ng synthetic somato- liberin 1 -44 (D-D) incubated at 4 °C for two days in 1 ml unextracted seminal plasma. Samples were eluted with radioimmunoassay buffer. Aliquots of the 1 ml fractions were assayed for Somatoliberin by radioim- munoassay. The column void volume (V0), total volume (Vt), and elution position of synthetic Somatoliberin are indicated by the arrows.
Somatoliberin 1-44
20 40
Elution volume [ml] 60 Fig. 3. Sephadex G-50 fine chromatography of
[125I]somatoliberin after two days incubation at 4 °C with either unextracted seminal plasma (α—α) or assay buffer (π —α). Samples were eluted with radioimmu- noassay buffer. The 1 ml fractions were directly counted in a gamma-counter. The column void volume (V0), total volume (Vt), and elution position of synthetic Somatoliberin are indicated by the arrows.
radioimmunoassay buffer was incubated with 0.5 ml unextracted seminal plasma at 4 °C for two days, in order to reproduce the conditions of our somatoli- berin radioimmunoassay. At the end of the incubation period the mixture was chromatographed on the Sephadex G-50 column, then fractions were measured in radioimmunoassay. Control experiments for the recovery of Somatoliberin standard following gel chromatography were performed by incubating 25 ng Somatoliberin standard in 1 ml radioimmunoassay buffer for two days at 4 °C before chromatography, and 25 ng Somatoliberin standard in 0.5 ml radioim- munoassay buffer mixed with 0.5 ml seminal plasma just before gel permeation. As shown in figure 2 only a trace amount of Somatoliberin standard was re- covered in the position where Somatoliberin standard eluted. Aside from a small peak near the void volume of the column, no other peak of somatoliberin-like immunoreactivity was found. The recovery of soma- toliberin standard incubated in radioimmunoassay buffer alone was 88.5%, whereas, after two days in- cubation in seminal plasma only 4.6% of added so- matoliberin standard was recovered at the correct elution position; on the other hand, immediate chro- matography, without incubation of Somatoliberin standard mixed with seminal plasma, resulted in a minimal loss of recovered Somatoliberin (71.9%). The anomalous, peak at the void volume of the column was still present.
To further investigate the nature of this nonspecific effect, Somatoliberin tracer and unextracted seminal plasma were incubated together at 4 °C for two days.
As shown in figure 3, this procedure resulted in an almost complete degradation of labelled somatoli- berin with generation of a new peak of radioactive material eluting shortly after the total volume of the column. Numerous attempts were made to eliminate this degradative enzymic activity, e. g. by addition of enzyme inhibitors to seminal plasma before incuba- tion with labelled Somatoliberin. Aprotinin, phenyl- methylsulphonyl fluoride, and bacitracin did not, however, prevent Somatoliberin tracer degradation.
Aliquots of unextracted seminal plasma were heated to 90 °C for 10 min, centrifuged to remove precipi- tated proteins, and subsequently measured in radioim- munoassay or incubated with labelled Somatoliberin.
Heat treatment reduced dramatically the amount of somatoliberin-like immunoreactivity of seminal plasma (0.8 μg/l). Moreover, Somatoliberin tracer was not degraded by coincubation with heated seminal plasma, as shown in figure 4. No radioactive peak eluted after the normal Somatoliberin peak; however, another anomalous peak appeared near the void vol- ume of the column (fig. 4), probably due to nonspe- cific binding of Somatoliberin tracer to denatured proteins. This suggestion was supported by the finding that prior ultrafiltration of the heated seminal plasma, using Centrisart I (Sartorius GmbH, G ttingen, Fed- eral Republic of Germany; exclusion limit: Mr
= 10000-15000), led to disappearance of the first peak of radioactivity (fig. 4). Ultrafiltered seminal plasma contained no detectable amount (< 50 ng/l) of somatoliberin-like immunoreactivity by radioim- munoassay.
J. Clin. Cbem. Clin. Biochem. / Vol. 26,1988 / No. 6
370 Losa et al.: Somatoliberin-like immunoreactivity in human seminal plasma: RIA interference
Somatoliberin
20000-
ΊΟ 000 aο
"•6
20 40
Edition volume [ml] 60
Fig. 4. Sephadex G-50 fine chromatography of [125I]somatoliberin after two days incubation at 4°C with either unextracted seminal plasma heated at 90 °C for 10 min (α —α) or unextracted seminal plasma heated at 90 °C for 10 min and subsequently ultrafiltrated (D —D). Samples were eluted with radioimmunoassay buffer. The 1 ml fractions were directly counted in a gamma-counter. The column void volume (V0), total volume (Vt), and elution position of synthetic somato- liberin are indicated by the arrows.
Discussion
Using a highly specific antibody and purified ra- dioiodinated somatoliberin, we developed a radioim- munoassay system able to detect 50 — 70 ng/1 soma- toliberin. When unextracted seminal plasma was as- sayed, high somatoliberin levels were found and di- lution curves of synthetic somatoliberin and unex- tracted seminal plasma ran almost completely parallel (fig. 1). However, we found that extraction of seminal plasma by affinity chromatography or adsorption onto CIS Sep-pak cartridges led to disappearance of somatoliberin-like immunoreactivity from seminal plasma, whereas such immunoreactivity was still pres- ent in the effluent after the adsorption step. The Chromatographie profile of seminal plasma demon- strated the presence of a major peak of somatoliberin- like immunoreactivity near the void volume of the Sephadex G-50 fine column and the complete disap- pearance of immunoreactivity from the elution posi- tion of somatoliberin standard (fig. 2). This peak near the void volume of the column might be due to aggregation forms of somatoliberin (at least 4-6 molecules, in view of the exclusion limit of the Seph- adex column), adsorption of somatoliberin onto sem- inal plasma proteins, or elution of degrading en- zyme(s), which in the somatoliberin radioimmunoas- say could cause destruction of the tracer, leading to a misleadingly high somatoliberin concentration. The latter hypothesis is supported by the experiment shown in figure 3. Incubation of seminal plasma with somatoliberin tracer led to an almost complete dis- appearance of the peak at the void volume of the
column, thus excluding aggregation of radiolabelled somatoliberin molecules or their binding to native seminal plasma proteins; it also resulted in the ap- pearance of nearly all radioactive material shortly after the total volume of the coltnnn, as if labelled somatoliberin were completely destroyed by an en- zymic degrading activity present in seminal plasma.
Elution of small fragments of somatoliberin shortly after the total volume of the column, though uncom- mon, is not inkown. Similar effects have been reported for other peptides. They seem to be due to interaction of the fragments with the column packing material (13, 14). These results demonstrate the non-identity of somatoliberin standard with the somatoliberin-like immunoreactivity of seminal plasma. This fact un- derscores the notion that parallelism between stand- ard and "unknown" in a radioimmunoassay system is a prerequisite, but it is not sufficient to validate the method.
Degradation of hypothalamic hormones by pepti- dases in rat and human serum has already been re- ported (15) and has been postulated to cause high
"blank effects" in one radioimmunoassay for soma- tostatin (16). Nevertheless, enzymic degradation of the tracer is probably an underestimated problem in validating a radioimmunoassay system (17).
Rafferty & Schulster (18) found the same degradation of labelled somatoliberin after overnight incubation with rat plasma. However, using a Sephadex G-50 column, they found that the smaller radiolabelled component(s) eluted before the total volume of the column. This discrepancy with our results can be best explained, apart from differences in the methodology, by the different somatoliberin preparation used in the two studies, namely somatoliberin 1—40 in Rafferty
& Schulster study (18) and somatoliberin 1-44 in ours. Confirming previous results (18), we were also unable to prevent somatoliberin tracer degradation by addition of effective concentrations of protease inhibitors like aprotinin, phenylmethylsulphonyl fluoride, and bacitracin. However, heating seminal plasma to 90 °C for 10 min resulted in loss of the degradative enzymic activity of seminal plasma. The appearance of another peak of radioactivity near the void volume of the column is probably due to non- specific adsorption of somatoliberin tracer to dena- tured proteins produced by the heating'procedure.
This suggestion is supported by the fact that when labelled somatoliberin is incubated with heated and subsequently ultrafiltered seminal plasma, gel per- meation chromatography shows p$ly one peak of radioactivity coeluting with somatoliberin standard.
Moreover, somatoliberin measurement in heated sem-
J. Clin. Chem, Clin. Biochem. / Voh 26, 1988 / No. 6
Losa et al.: Somatoliberin-like immunorcactivity in human seminal plasma: RIA interference 371 inal plasma demonstrated a dramatic reduction of
somatoliberin-likc immunoreactivity. The minimal re- sidual somatoliberin activity is very probably due to nonspecific binding of somatoliberin to denatured proteins and not to residual degradative enzymic ac- tivity. Very recently, Frohnan and coworkers (19) described the rapid enzymic degradation of somato- liberin to a biologically inactive product by plasma both "bi vivo" and "in vitro" consisting of the removal of two amino acids from the NH2-terminus. The en- zyme(s) responsible has not yet been identified, though a dipeptidylaminopeptidase has been sug- gested (19). It'is tempting to speculate that the same degradative enzymic activity is present in seminal plasma; since the first amino acid of somatoliberin is tyrosine, a radioiodinated dipeptide should elute near the total volume of the Sephadex G-50 column. How- ever, somatoliberin contains a second tyrosine residue
at position 7. If, therefore, the cleavage of the two first amino acids at the NH2-terminus were the only enzymic degrading activity of seminal plasma, we should observe a second peak of radioactivity coelut- ing near the intact somatoliberin standard and cor- responding to labelled somatoliberin 3 — 44. It seems likely that degradation of somatoliberin by seminal plasma is more complete and probably leads to pro- duction of different small fragments.
In conclusion, our study demonstrates that somato- liberin-like immunoreactivity in seminal plasma is an artifact due to degradative enzymic activity, which is able to destroy radiolabelled and, probably, native somatoliberin. It is suggested that coincubation stud- ies of labelled substances and organic fluids must be performed in order to reveal tracer degradation as a source of misleading results in radioimmunoassays.
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Dr. Klaus von Werder
Medizinische Klinik Innenstadt Universität München
Ziemsscnstr. l D-8000 München 2
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